Enzymatic Reaction Mechanisms
We will study the reaction mechanisms of chymotrypsin,
hexokinase, and enolase because they are some
of the best understood enzymes and they illustrate some
general principles of enzymatic reaction mechanisms.
Chymotrypsin is a protease that catalyzes hydrolysis
of _________ bonds adjacent to __________ residues.
Hydrolysis of peptide bonds is thermodynamically _________ (negative ∆G),
but the reaction is very _______.
Chymotrypsin enhances the rate by a factor of ~109.
Composed of 3 polypeptide chains, linked by disulfide bonds.
Active-site residues (in red) are far apart in the _________ sequence.
Fig 6-18
1
Chymotrypsin structure
The active-site residues His57,
Asp102, and Ser195 (red)
are close in _________ structure.
red = ____________ residues
green = pocket where aromatic
side chain of __________ binds
Fig 6-18
A close-up of the active site of chymotrypsin
green = substrate
(polypeptide chain)
blue = aromatic
side chain of
substrate in its
binding pocket
red = activesite residues
purple = carbonyl
of aromatic residue
Asp102 is down here
Fig 6-18
2
Chymotrypsin’s mechanism
Follow along on the handout (Lehninger p. 216-217). An example of
general acid catalysis, general base catalysis, and covalent catalysis.
____________: His57, Asp102, Ser195
linked together in a _________-bonding
network
Ser195 is deprotonated by general base catalysis (_______ is the base).
Ser has very high pKa; His57 has pKa ____ due to the H-bonding network.
(alkoxide ion:
when H+ is
removed from
hydroxyl of
Ser195)
backbone amide N
of Ser 195
3
2.
1. The short-lived
tetrahedral intermediate
is shown here; its neg.
charge is stabilized
by two backbone
amide protons.
general acid catalysis
by His57
stabilization of
negative charge
polypeptide has been
cleaved on __________
side of aromatic residue
The peptide bond has now been cleaved by the enzyme, but the aromatic
residue of the peptide is covalently bound to Ser195 at this point. This is
an example of __________ catalysis (a ________ covalent bond is formed
between the enzyme and the substrate).
4
His57 is again responsible for general base catalysis:
carboxylate
______ is responsible for general acid catalysis– transfers proton to _______
5
His57, Asp102, Ser195 are
back to their original states,
ready for another round of
catalysis.
Catalysts, including enzymes, are not changed
or used up in a chemical reaction.
Chymotrypsin belongs to a family of enzymes called _________________,
which all have the essential active-site __________ residue.
Other serine proteases: trypsin (cleaves on C-terminal side of Lys, Arg)
elastase
Understanding general acid-base catalysis:
View Fig 6-9 on page 201 to see all the amino acid side chains that
can participate in general acid-base catalysis.
Be sure you understand why and how each residue can participate (they
each donate or accept protons, like chymotrypsin’s mechanism).
Be sure you understand which forms participate in general base
catalysis and which participate in general acid catalysis.
6
Hexokinase is a bisubstrate enzyme
responsible for phosphorylation of glucose
substrates: glucose and Mg·ATP
The reaction:
page 218
Water molecules are present in the active site and are similar in reactivity
to the __________ group of glucose.
How does hexokinase distinguish between glucose and water as
the substrate for phosphorylation?
Hexokinase has an
not positioned for reaction.
conformation where active site residues are
When the correct substrate (glucose) binds to its binding site, the binding
energy provides energy for hexokinase to change ________________ to
an
form where active-site residues are properly positioned.
Binding of water will not cause this conformational change.
This is an example of ____________ (binding of the substrate induces the
enzyme to change conformations so it fits the substrate.)
glucose
binding
Fig 6-22
conformational
change
7
Enolase illustrates metal ion catalysis
Dehydration of 2-phosphoglycerate to phosphoenolpyruvate
Mg2+ makes proton
more _______ (lower pKa)
intermediate stabilized
by Mg2+
general ______ catalysis
by Lys345
general ______ catalysis
by Glu211 to generate
a good leaving group
Fig 6-23
(Ch. 6.3) Enzyme activity depends on pH because:
1. side chains in active site must maintain a certain state of __________ in
order to act as weak acids or bases
2. ionized side chains throughout the structure play a role in maintaining
the __________ structure of the protein
Fig 6-17
hydrolyzes peptide bonds
found in liver cells; releases
of proteins during digestion
glucose into blood stream
in stomach
#18 (p. 237) recommended!
8
Regulatory Enzymes
Chapter 6.5
Lehninger, page 232:
“We began this chapter by stressing the central importance of catalysis
to the very existence of life. The control of catalysis is also critical to
life. If all possible reactions in a cell were catalyzed simultaneously,
macromolecules and metabolites would quickly be broken down to
much simpler chemical forms. Instead, cells catalyze only the reactions
they need at a given moment.”
How do cells control the rates and timing of catalysis by enzymes?
Groups of enzymes work together in sequential
pathways during cellular metabolism.
Example shown where _________ of one
enzymatic reaction is __________ of next.
______________ enzyme– one enzyme in the
pathway that controls the rate of the overall
sequence of reactions.
The rate of the regulatory enzyme is adjusted
(increased or decreased) in response to certain
signals.
When the rate of the regulatory enzyme
changes, the rate of the __________________
changes.
Fig 14-11
9
The rates of regulatory enzymes are modulated in 4 ways:
1. (allosteric enzymes only) reversible, noncovalent binding of
allosteric modulators (small metabolites or cofactors)
2. reversible ___________ modification
3. binding of another protein
4. irreversible removal of peptide segments by _____________________
We will discuss 1, 2, and 4 in more detail.
1. Allosteric regulatory enzymes undergo _______________
changes in response to binding of an allosteric modulator
(reminder: allosteric proteins are those that change conformation upon
binding certain molecules)
R = regulatory subunit
C = catalytic subunit
Note: S and M have
different binding sites
(most allosteric enzymes
have 2 or more subunits)
Sometimes the Modulator
is actually the Substrate
itself
conformational change
due to communication
between subunits
rate of catalysis
increases; rate of entire
pathway increases
Fig 6-26
10
Allosteric regulatory enzymes
may be modulated by the end
product of the pathway: called
.
Example:
Threonine dehydratase is inhibited
allosterically by L-isoleucine (its ___________
and the end product of the pathway).
When L-isoleucine builds up, it binds to
the regulatory site on threonine dehydratase
(not the active site). The binding is noncovalent and reversible.
Build up of L-isoleucine slows the rate of
catalysis by threonine dehydratase, and
the ________________ is slowed as a result.
When L-isoleucine concentration decreases,
it dissociates from the enzyme and the rate of
the pathway
.
Fig 6-28
2. Some regulatory enzymes undergo
reversible covalent modification
Most common:
1/3 - 1/2 of
eukaryotic proteins
are phosphorylated.
Addition of one of these
groups to an enzyme
causes the enzyme to
become more active, and
the rate of the whole
pathway increases.
Fig 6-30
11
_____________________and__________ side chains can
be phosphorylated on their hydroxyl groups
_________– enzymes that catalyze the covalent attachment of phosphoryl
groups to these side chains on other proteins
_________________– enzymes that catalyze the removal by hydrolysis of
phosphoryl groups from these side chains on other proteins
Fig 8-40
Fig 6-30
Phosphoryl group comes from ATP (adenosine triphosphate).
Phosphoryl group is bulky and __________ charged; can H-bond with other
groups, interact favorably with Arg or Lys, repel Asp or Glu.
Thus: phosphorylation can have dramatic effects on protein _____________.
Phosphorylation can cause conformational changes in
an enzyme, which results in increased or decreased
substrate binding and/or enzymatic activity.
Example:
addition of a phosphoryl group causes a
_____________ change,
including a change in
the active site structure
Fig 6-31
12
4. Some enzymes are regulated by irreversible
proteolytic cleavage of an enzyme precursor
An ________ precursor protein is cleaved to form the _______ enzyme
Fig 6-33
(A, B, C linked by disulfide bonds)
Cleavage causes a conformational
change that exposes _____________
residues.
The enzyme must be inactivated by
another mechanism, such as
inhibitor binding to active site.
An example of enzyme regulation that will be
familiar from Chem 431 lab: mushroom tyrosinase
Tyrosinase is an enzyme in plants and fungi that catalyzes a reaction
which results in browning of the tissue when the organism is damaged.
Tyrosinase exists in an inactive form with two domains. The N-terminal
domain contains the active site. The C-terminal domain forms a
protective cap over the active site.
When the organism is damaged, a signal (maybe phosphorylation)
instructs proteases in the cell to cleave off the C-terminal domain. This
exposes the active site, so tyrosinase becomes active and catalyzes the
browning reaction.
cleavage here
pink = N-terminal domain
green = C-terminal domain
(structure shown is for a
phosphorylation here?
protein that is similar to
mushroom tyrosinase)
active site here (protected
by C-terminal domain)
13